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Uterine spiral artery remodeling is required for successful human pregnancy; impaired remodeling is associated with pregnancy complications, including late miscarriage, preeclampsia, and fetal growth restriction. The molecular triggers of remodeling are not known, but it is now clear that there are "trophoblast-independent" and "trophoblast-dependent" stages. Uterine natural killer (uNK) cells are abundant in decidualized endometrium in early pregnancy; they surround spiral arteries and secrete a range of angiogenic growth factors. We hypothesized that uNK cells mediate the initial stages of spiral artery remodeling. uNK cells and extravillous trophoblast (EVT) cells were isolated from early pregnancy decidua and placenta. Chorionic plate arteries from full-term placentas and spiral arteries from nonpregnant myometrium were cultured with angiogenic growth factors or conditioned medium (CM) from uNK cells or EVT or uNK cell/EVT cocultures. In both vessel models, uNK cell CM induced disruption of vascular smooth muscle cells (VSMCs) and breakdown of extracellular matrix components. Angiopoietin (Ang)-1, Ang-2, interferon-γ, and VEGF-C also disrupted VSMC integrity with an Ang-2 inhibitor abrogating the effect of uNK cell CM. These results provide compelling evidence that uNK cells contribute to the early stages of spiral artery remodeling; failure of this process could contribute to pregnancy pathology.

During human uterine spiral artery (SpA) remodeling, vascular smooth muscle cells (VSMCs) are lost and replaced by fibrinoid, incorporating extravillous trophoblast (EVT) cells. The aim of the current study was to determine the relative contributions of apoptosis and migration to VSMC loss during SpA remodeling. Immunohistochemistry (Apoptag, active caspase 3, lamin) of placental bed biopsies (8-20 wk gestation) demonstrated apoptotic cells in all samples; double immunolabeling identified these as trophoblasts, leukocytes, and endothelial cells. In total, 294 SpAs were studied, and only one apoptotic VSMC was identified. H-caldesmon-immunopositive VSMCs were observed surrounding and separate from SpA walls in partially remodeled vessels; the highest level of VSMC migration was observed in vessels with associated EVT cells (number of migrated cells 6.4 ± 1.2; distance migrated 3.5 ± 0.3 pixels) compared with those without (number of migrated cells 3.6 ± 0.5, P<0.001; distance migrated 2.8 ± 0.1 pixels, P<0.0001). VEGF-A, VEGF-C, TGF-β1, and Ang-2 all stimulated human aorta VSMC invasion in vitro, although EVT cell culture supernatants did not. In summary, apoptosis is unlikely to play a major role in loss of VSMCs from SpAs during remodeling in normal pregnancy, but VSMCs appear to migrate away from the wall of the SpA, an effect enhanced by the presence of EVT cells.

Background: Extravillous trophoblast cell (EVT) invasion of decidua and inner third of the myometrium is critical for a successful pregnancy. Many decidual factors are likely to play a role in regulating this process, including uterine natural killer (uNK) cell-derived cytokines., Hypotheses: 1) uNK cells are a major source of IFN gamma (IFN-gamma) and 2) IFN-gamma inhibits EVT invasion via an increase in EVT apoptosis and/or a decrease in active protease levels., Methods: Total decidual and uNK cells from 8-10 wk and 12-14 wk gestational age were cultured. IFN-gamma mRNA (real-time RT-polymerase chain reaction) and protein levels (FastQuant multicytokine analysis) were determined. EVT invasion in the presence of IFN-gamma or anti-IFN-gamma-neutralizing antibodies was assessed. Trophoblast apoptosis and proliferation was assessed in explants by immunohistochemistry for M30 and Ki67. Substrate zymography was performed to determine levels of secreted MMP2, MMP9, and uPA., Results: mRNA and protein for IFN-gamma was detected in both total decidual and uNK cell fractions. Trophoblast invasion was inhibited by IFN-gamma. The level of M30-positive EVT was increased in the presence of IFN-gamma whereas levels of secreted MMP2 were decreased., Conclusions: uNK cells are a source of IFN-gamma within early human pregnancy decidua. Mechanisms of IFN-gamma inhibition of EVT invasion include both increased EVT apoptosis and reduced levels of active proteases.

Human securin, known also as PTTG, has established oncogenic and cell cycle regulatory functions. PTTG/securin transforms cells in vitro, inhibits sister chromatid separation, and regulates secretion of fibroblast growth factor-2. FGF-2 is a key regulator of CNS development and PTTG/securin expression has been reported in murine fetal brain. We examined the expression and function of securin and FGF-2 in the developing human fetal brain and in a fetal neuronal cell line (NT 2). Securin expression was significantly reduced in first and second trimester fetal cerebral cortex compared with adult cerebral cortex, where immunocytochemistry revealed intense securin staining in neuronal cell bodies. FGF-2 protein was concordantly lower in fetal cortex, whereas pretranslational expression of PTTG binding factor (PBF) was not significantly altered in fetal brain compared with adult. PCNA expression demonstrated that high securin levels in adult cortex were associated with absent cell proliferation. In NT-2 cells, securin stimulated FGF-2 expression, which could be abrogated by a carboxyl-terminal mutation. Low transient expression of securin resulted in a significant proliferative effect, whereas high levels of securin expression inhibited cell turnover. We propose a potential role for human PTTG/securin in modulating cell proliferation and FGF-2 expression during human neurogenesis.

In this study we tested the hypothesis that expression of heme oxygenases HO-1 and HO-2, which are responsible for the production of carbon monoxide, are reduced in the placenta and placental bed of pregnancies complicated by preeclampsia (PE) and fetal growth restriction (FGR) compared with control third-trimester pregnancies. Placental protein expression was determined by Western blotting (n=10 in each group) and immunohistochemistry (controls n=18, PE n=19, FGR n=10). Extravillous trophoblast expression was determined by immunohistochemistry of placental bed biopsy samples (controls n=17, PE n=19, FGR n=10). Western blot analysis of placental homogenates showed no overall differences in HO-2 among groups. However, immunohistochemical analysis showed a reduction in HO-2 expression in endothelial cells in both abnormal groups (PE P<0.01; FGR P<0.0005 vs. control group) but no differences in villous trophoblast staining. HO-1 was undetectable by Western blotting in control and abnormal pregnancies and immunoreactivity was very low, suggesting that there is little HO-1 in the placenta. Within the placental bed, HO-2 but not HO-1 was detected on all populations of extravillous trophoblast, but expression of HO-2 or HO-1 did not change in PE or FGR. The reduced expression of HO-2 on endothelial cells in PE and FGR may be responsible for reduced placental blood flow in these conditions. The data do not show changes in HO in the placental bed in PE or FGR.

The purpose of this study was to examine the expression of hemeoxygenases HO-1 and HO-2, which are responsible for the production of carbon monoxide (CO), in the human placenta and placental bed and to determine the role of inhibitors of HO on placental perfusion pressure. We hypothesized that HO is expressed within the placenta and that invading cytotrophoblast cells (CTB) express HO isoforms. The expression of HO-1 and HO-2 was studied on placenta and placental bed biopsies, obtained using a transcervical sampling technique, from normal human pregnancies between 8 and 19 wk gestation and at term. In the placenta, HO-2 immunostaining was prominent in syncytiotrophoblast in the first trimester and reduced toward term (P<0.0005). HO-2 endothelial immunostaining was weak in the first trimester, but increased by term (P<0.0005). Within the placental bed, HO-2 was expressed by CTB in cell columns, the cytotrophoblast shell, and cell islands. Both intravascular CTB and interstitial CTB expressed HO-2. HO-1 immunostaining was low in the placenta but intense on the CTB within the placental bed. A striking feature was the absence of HO-1 from the proximal layers of cell columns, with strong expression on the more distal CTB layers of the cell columns. In placental perfusion studies, a significant dose-dependent increase in perfusion pressure was observed in the presence of zinc protoporphyrin, an inhibitor of HO. These results suggest a role for CO in placental function, trophoblast invasion, and spiral artery transformation. Hemeoxygenase expression in human placenta and placental bed implies a role in regulation of trophoblast invasion and placental function.